Eight fatty acids (C12–C18) were previously identified in human amniotic fluid, colostrum, and milk in similar proportions but different amounts. Amniotic fluid is well known to be the natural environment for development in mammals. Interestingly, amniotic fluid and an artificial mixture of fatty acids contained in amniotic fluid produce similar anxiolytic-like actions in Wistar rats. We explored whether the lowest amount of fatty acids contained in amniotic fluid with respect to colostrum and milk produces such anxiolytic-like effects. Although a trend toward a dose-response effect was observed, only an amount of fatty acids that was similar to amniotic fluid fully mimicked the effect of diazepam (2 mg/kg, i.p.) in the defensive burying test, an action devoid of effects on locomotor activity and motor coordination. Our results confirm that the amount of fatty acids contained in amniotic fluid is sufficient to produce anxiolytic-like effects, suggesting similar actions during intrauterine development.
Fatty acids (FATs) are organic lipid compounds that consist of a hydrocarbon chain of variable length and carboxyl group at the end of the chain [
We recently reported that human amniotic fluid, colostrum, and milk consistently contained eight FATs (C12:0, lauric acid; C14:0, myristic acid; C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1
Earlier observations indicated that olfactory stimulation with maternal amniotic fluid or maternal milk decreased grimaces and crying in human newborns when their mothers were absent [
Because amniotic fluid may exert multiple functions [
All of the experiments were performed in accordance with international ethical standards based on the Guide for the Care and Use of Laboratory Animals [
Seventy-one adult male Wistar rats were obtained from a local strain initially supplied by Harlan (Mexico City, Mexico). The rats were housed in local housing facilities at a mean temperature of
A mixture of eight FATs was prepared according to previous studies [
An acrylic box (27 × 17.5 × 15.5 cm) with the floor covered by a 5 cm bed of fine sawdust (Teklad Sani-Chips 7090, 2.2 cubic feet; Harlan, Indianapolis, IN, USA) was placed inside a noise-isolated box (65 × 55 × 45 cm; Coulbourn Instruments, Whitehall, PA, USA). An electrode (7 cm length, 0.5 cm diameter) protruded 2 cm above the sawdust bed horizontally from one wall of the box (17.7 × 15.5 cm). The electrode delivered a constant-intensity direct current (0.3 mA) from an electronic stimulator (Grass Instruments S44, Quincy, MA, USA) coupled in series to a stimulus isolation unit (Grass Instruments SIU5) and constant-current unit (Grass 7 Instruments CCUIA). When a rat incidentally touched the electrode, it received an electric shock and began to vigorously displace the sawdust to cover the electrode. All of the sessions were recorded for subsequent analysis by two independent observers to measure the burying latency and cumulative burying time during a 10 min test. After each test session, the bed of fine sawdust was removed and replaced by clean sawdust bedding. Only observations with more than 95% agreement between observers were included in the data analysis. Immediately after the defensive burying test, each rat was evaluated in the open field test.
To evaluate the effects of the treatments on spontaneous locomotor activity, the rats were subjected to a 5 min open field test. We used an automated motor activity monitor (Acti-Track v2.7.10, PanLab, S.L. Instrument, Barcelona, Spain) in a perspex box (45 × 45 cm base, 35 cm height). A total of 32 infrared beams, 16 each on perpendicular walls, were mounted 3 cm above the box frame floor and connected to an interface (LE 8811, LSI Letica Scientific Instruments, Barcelona, Spain). The data were sent to a computer. For data analysis, the floor of the cage was divided into five equally sized virtual squares (four peripheral squares and one central square), and we measured the total number of entries into the squares (i.e., crossings), time spent active (in seconds), and time spent resting (in seconds).
After each experimental session, the open field box was carefully cleaned and deodorized with a cleaning solution (0.5% ammonia, 15% ethanol, 10% extran, 5% isopropyl alcohol, 19% Pinol, and 50.5% water). Five minutes elapsed between each test to allow the scent of the substances to dissipate.
To assess the effects of the treatments on motor coordination, all of the rats underwent a 3-day training program on a 7-cm diameter rotarod (LE 8300, LSI Letica, Panlab Scientific Instruments, Barcelona, Spain). During the training period, each rat was placed on a horizontal rod that rotated at a gradually increasing speed of 4 to 20 rotations per minute for a maximum period of 5 min to determine baseline performance. The day after training, motor coordination was recorded by gradually increasing the speed from 4 to 20 rotations per minute during five trials with a cutoff time of 3 min. The dependent variable was the total time spent on the rotating rod (in seconds).
We used a transversal design, with six independent groups. One group received vehicle (1.0 mL/rat, s.c.;
Fatty acid concentrations contained in volume administered to each rat. The fatty acid concentrations contained in 1 mL of the artificial FAT mixture correspond to the proportion found in human amniotic fluid [
Fatty acid | Volume | ||||
---|---|---|---|---|---|
0.125 mL | 0.25 mL | 0.5 mL | 1 mL | Content ( |
|
C12:0 (Lauric) | 0.5 | 1.0 | 2.0 | 4.0 | 0.9 |
C14:0 (Myristic) | 3.7 | 7.5 | 15.0 | 30.0 | 6.9 |
C16:0 (Palmitic) | 19.1 | 38.2 | 76.5 | 153.0 | 35.3 |
C16:1 (Palmitoleic) | 8.8 | 17.7 | 35.5 | 71.0 | 16.4 |
C18:0 (Stearic) | 4.6 | 9.2 | 18.5 | 37.0 | 8.5 |
C18:1 |
10.0 | 20.0 | 40.0 | 80.0 | 18.4 |
C18:1 |
1.8 | 3.7 | 7.5 | 15.0 | 3.5 |
C18:2 (Linoleic) | 5.5 | 11.0 | 22.0 | 44.0 | 10.1 |
| |||||
Total | 54 ( |
108.3 ( |
217 ( |
434 ( |
100 |
After the anxiety test, other groups of rats were subjected to a transversal design, with three independent groups. One group received vehicle (1.0 mL/rat, s.c.;
The data were analyzed using one-way analysis of variance (ANOVA) for independent groups. Values of
The burying latency analysis revealed significant differences among treatments
Burying latency. A volume of 1 mL of the artificial FAT mixture and diazepam (2.0 mg/kg) significantly (
The analysis of cumulative burying time also revealed significant differences among treatments
Cumulative burying. Cumulative burying time was significantly less (
The analysis of locomotor activity did not detect significant differences between peripheral and central activity in any of the measures. Therefore, we analyzed the total values of these variables during the 5 min test (Table
Effect of treatment in open field test. The data are expressed as mean ± standard error of the mean. No significant differences in activity time, resting time, or crossings were found between groups.
Group | Time active (s) | Time resting (s) | Crossings ( |
---|---|---|---|
Vehicle (1 mL) | 164.8 ± 7.85 | 135.2 ± 7.85 | 98.4 ± 13.69 |
Artificial fatty acid mixture | |||
0.125 mL | 149.6 ± 14.05 | 150.4 ± 14.05 | 92.8 ± 16.01 |
0.25 mL | 133.2 ± 8.34 | 166.8 ± 8.34 | 74.0 ± 10.18 |
0.5 mL | 160.5 ± 13.80 | 139.5 ± 13.80 | 86.8 ± 16.09 |
1 mL | 152.5 ± 14.69 | 147.5 ± 14.69 | 85.0 ± 12.14 |
Diazepam (2 mg/kg) | 117.8 ± 18.15 | 182.2 ± 18.15 | 58.2 ± 11.85 |
The analysis of the total time on the rotarod did not detect significant differences between the groups treated vehicle
The aim of the present study was to determine whether the amount of FATs contained in amniotic fluid produces anxiolytic-like effects on rats in the defensive burying test. The results demonstrated that only the same amount of FATs found in amniotic fluid, but not lesser amounts, mimicked the anxiolytic-like effects of diazepam, without altering spontaneous locomotor activity or motor coordination.
Other studies demonstrated the anxiolytic-like effects of FATs in humans [
In the defensive burying test, the time that elapsed between the first shock and first attempt at burying (i.e., burying latency) is inversely related to the rat’s reactivity [
Cumulative burying time in the defensive burying test is considered an index of reactive anxiety produced by electric shock. Longer cumulative burying times purportedly reflect greater levels of anxiety [
We found a statistically significant dose-response relationship [
Changes in locomotor activity may interfere with performance in the defensive burying test. Therefore, the measure of locomotor activity is commonly used to exclude putative nonspecific drug effects in the defensive burying test [
Other behavioral studies on the anxiolytic-like effects of FATs did not include active controls in humans [
The authors declare that there is no conflict of interests.
The authors thank Michael Arends for revising and editing the English of this manuscript. This study was partially supported by a grant from the Consejo Nacional de Ciencia y Tecnología, México (CONACyT: CB-2006–1, 61741) and DGAPA-PAPIIT IN211111-3 from the Universidad Nacional Autónoma de México, who did not participate in the study design, data collection, data analysis, interpretation of the data, writing of the manuscript, or decision to submit the paper for publication. The first author received fellowships from CONACyT (Reg. 203280).